Seeds used in the present study were collected in the spring of 2013 from 29 wheat fields infected with rigid ryegrass and located in the southwestern part of Iran (Khuzestan province) (Table 1). In the specific area, there were often complains on the low efficacy of ACCase herbicides, while there were several reports of herbicide resistance development. Seeds were mainly collected at the beginning of maturity from herbicide-treated wheat fields along with some fields that had never been treated with herbicides (in order to use them as susceptible). Each surveyed field was walked through by the two diagonals seeds were separated, air-dried, and stored at room temperature (22–26°C) until their use. Rigid ryegrass seeds were treated with 10 ppm of gibberellic acid (GA) at 5 days before sowing, in order to relieve any seed dormancy.

Ten germinated seeds of the potentially resistant and susceptible populations were sowed in pots filled with 500 ml of soil. An herbicide-free soil mixed with a common peat substrate (1:1, V/V) was used. Throughout the experiments the pots were uniformly watered as needed. Shortly after emergence, plants were thinned to a final density of seven seedlings per pot. The pots were fertilized as required and kept under conditions set at 20–30/10–15°C day/night with a 14 h photoperiod and a relative humidity of about 65%. The required amount of light was provided by a combination of fluorescent and incandescent lamps. At 21 DAE¹ (at the 2–3 leaf stage), seedlings were treated with the recommended dose of clodinafop-propargyl (CLD) and pinoxaden (PIN). Particularly, CLD and PIN were applied at rates of 64 and 45 g ai ha⁻¹, respectively. Plants were harvested at 28 DAT² and the fresh weight of foliage was recorded, with the number of dead and surviving plants also being measured.

After the above-mentioned evaluation of efficacy and potential resistance, the populations were used for leaf sampling and subsequent DNA extraction. Particularly, in a CAPS (cleaved amplified polymorphic sequences) analysis, gene-specific primers were applied to reproduce DNA pattern, and SNPs (single nucleotide polymorphisms) were identified. In the dCAPS (Derived cleaved amplified polymorphic sequences) technique, a limitation enzyme identification site, which contains the SNP, was introduced into the PCR amplicon by a primer containing one or more inconformities to the DNA template. The PCR amplicon is then digested by a limitation enzyme, and the presence or absence of the SNP is defined by the resulting digestion restriction template as previously described by Yu et al. (2008).

In this study, the effect of sowing depth on emergence of three rigid ryegrass populations (a susceptible population and two resistant populations) was evaluated. Mean seed weight (MSW) was similar for the three populations (MSW of 1.85 ± 0.25 mg, n = 100) in order to eliminate any side effects. Twenty seeds of each population were sown in 12-cm diameter pots at five depths (0, 2, 4, 6, and 8 cm). Pots were placed in a controlled environment greenhouse with the same conditions as above. The experiment was set up under a completely randomized experimental design with four replicates. Seedling emergence was measured daily for 21 days, while in the case of 0 cm, seedlings were considered emerged when the coleoptile length was 3 mm long (Steadman et al., 2003).

Fifteen seeds of each population were sown in a potting soil compound in 12-cm diameter pots. Pots were placed in a controlled environment greenhouse with the same conditions as above and watered uniformly as needed. Shortly after emergence, plants were thinned to a final density of eight seedlings per pot. The experiment was set up under a completely randomized experimental design with four replicates. The experiments were established in October 2014 and 2015. Four wheat and rigid ryegrass plants were harvested at 20-days intervals, from 20 to 160 DAE. Plants were cut in the soil surface and height was also measured. The plants were dried at 74°C for 72 h and dry weight was determined.

These tests were conducted in order to evaluate the competitive ability, growth, and seed production of S and R rigid ryegrass populations and wheat under competitive and non-competitive conditions. For that purpose, pot experiments were conducted in the greenhouse of Agricultural Research Center of Khuzestan province, Iran. Seedlings (after seed placement in Petri dishes and their germination) were transplanted into 12-cm diameter pots filled with 500 ml of a manure-loam-sand mixture in 1:1:1 ratio. Competition between rigid ryegrass populations and wheat was studied by means of a replacement series experiment at five proportions (100:0, 75:25, 50:50, 25:75, and 0:100) at a fixed density of 8 plants (ryegrass/wheat) per pot. The pots were kept under conditions set at 20–30/10–15°C day/night with a 14-h photoperiod and watered uniformly as needed. The required amount of light was provided by a combination of fluorescent and incandescent lamps. The experiment was set up in a completely randomized experimental design with four replicates. The replacement series experiment was established on October 2014 and repeated on April 2015. The plants were dried at 74°C for 70 h and weighed. Plants from each species were separated into shoots and spikes, while plant height was also measured at harvest.

Differences between seed populations were evaluated after data expression as percentage of the untreated control. The percentage of surviving plants was also recorded at 28 DAT.

In order to evaluate the effect of burial depth on the emergence of resistant and susceptible populations regression and curve fitting is considered as a recommended type of analysis. A functional three parameter sigmoid (S) model using nonlinear least squares regression (Curve Fitter) was performed by means of the software SigmaPlot 12 (Systat Software, Inc.) and shown below:

In Equation (1), y is whole appearance (emergence) on time x, a is the highest emergence (percentage), t_E50 is the time to attain 50% of ultimate seedling emergence (day) and b represent the slope around t_E50. Emergence values were angular transformed. Parameter estimates were compared by one-way ANOVA and means were separated using least significant difference (LSD) (α = 5%).

A similar procedure as the one mentioned above was also followed for the evaluation of competitive ability, growth, and seed production of S and R populations under competitive and non-competitive conditions. Data on plant height and dry weight were subjected to ANOVA. Total shoot dry weight (SDW) was compared with the theoretical yields for equal competitive ability using 95% confidence interval (CI). For this statistical analysis SAS software was used (SAS Institute, 1987).

Assessment of herbicide resistance was conducted by means of the rating system given in Figure 1 as described by Moss et al. (1999). The results showed that L. rigidum fresh weight and survival were significantly different between populations at 28 DAT with CLD. Populations AH3, AH4, BOS1, BOS2, BOS5, DA1, DA2, HAM1, HAM2, HAM3, HAM4, HAM5, HAM6, HAM7, and KHO showed the lower fresh weight reduction, while other populations were adequately controlled by CLD.

These differences between the several potentially R populations indicate that inadequate control of ryegrass in other locations may be attributed to other reasons like an improper application time or method and not per se to herbicide resistance. The results of our screening tests showed that 10 populations of L. rigidum have resistance to CLD (34.5% of the total). After confirming the resistance of the populations to this herbicide, they have been used for leaf sampling for DNA extraction as described above.

Leaves of these plants were analyzed by CAPS and dCAPS methods for identification of possible mutations occurring in 2088, 2078, 2041, and 1781 locations of ACCase enzyme coding gene which is involved in the resistance of L. rigidum to ACCase inhibitors. Finally, there have been recognized two mutations (Ile 1781Leu and Ile 2041Asn) and it was found that the mutation 2041-Asn confers sufficient level of resistance to clodinafop-propargyl. Molecular analyses of resistance confirmed that the Ile 1781 Leu and Ile 2041 Asn mutations in the resistant biotypes endowed resistance to FOPs, but not to DIMs and DEN.

Our results showed that when seeds were placed on the soil surface (0 cm) and in a depth of 2 cm, the S biotype had slightly higher seed germination percentage than the two R populations (2041-Asn and 1781-Leu), but this difference was not statistically significant (Figure 2A and Table 2). In the case of seeds placed on soil surface, seedlings of the R-2041 population had the lowest appearance (83%) compared with populations R-1781 and S (88 and 91%, respectively). When the seeds were sown in a depth of 2–4 cm, R-1781 biotype systematically exhibited lower emergence than S and R-2041 biotypes (Figures 2B,C and Table 2). On the contrary, seeds sown in a depth of 6 cm had a germination percentage of 60, 61.25, and 62% for R-2041, R-1781, and S biotypes, respectively. This finding indicates no-significant difference in the percentage of germination (parameter a) between the three biotypes at this depth (Figure 2D and Table 2). However, when seeds were sown in a depth of 8 cm, final percentage of emerged seedlings was similar for R-2041 and R-1781, while both biotypes had significantly higher emergence than the S biotype (Figure 2E and Table 2). A functional three parameter sigmoid (S) model (equal) presented a significant fit (P < 0.001, R² = 0.98–0.99) to emergence data for all combinations of population and sowing depth. Values of t_E50 were increased with sowing depth (Table 2). R-1781 population exhibited significantly higher t_E50 values than the S and R-2041 populations at 0 and 2 cm depths, while R-2041 population had the highest values at depths of 4, 6, and 8 cm (Table 2).

In all three populations, increasing depth resulted to an emergence reduction. It has also to be noted that at the depth of 0 cm, the highest germination rate was that of S population (Table 2). Some of these findings are in accordance with similar results obtained in the study of Vila-Aiub et al. (2015b).

The results of biomass and plant height data fit to a functional three parameter sigmoid curve for the populations R-781, R-2041, and S under non-competitive conditions as given in Figure 3. The highest biomass and plant height were reached at 80 day after sowing (DAS). Our results showed that biomass of the R-2041 and R-1781 populations was similar to that of the S population. Regarding the maximum dry weight, there were no significant differences (p > 0.05) between the resistant and susceptible populations. As anticipated, the data followed a typical sigmoid growth pattern. Similar results have been found by other researchers on resistant and susceptible populations of weeds like kochia and downy brome (Thompson et al., 1994; Christoffoleti et al., 1997; Park et al., 2004). Concerning plant height, our results revealed some significant differences between the rigid ryegrass populations with S population being significantly higher than R-1781 population (Figure 3). These findings are not in full agreement with results of previous studies (Thompson et al., 1994; Christoffoleti et al., 1997; Park et al., 2004; Menchari et al., 2008).

Seed production of the three studied populations was also within a wide range. As shown in Table 3, R-2041 was the population with highest seed production, 19 and 27% higher than the corresponding value for S and R-1781 population, respectively. However, regarding thousand seeds' weight (TSW) there were not significant differences between the several populations.

Previous studies on several weeds have shown either higher seed production for R compared with the S population (Purrington and Bergelson, 1997; Park et al., 2004), or no differences at all between the several biotypes (Alcocer-Ruthling et al., 1992; Thompson et al., 1994; Travlos, 2013).

Evaluation of the competitive ability of R and S L. rigidum populations showed that increasing rigid ryegrass density from 2 to 6 plants pot⁻¹ reduced wheat height compared to the non-competitive conditions (Figures 4A,B). The results showed that interspecific competition (rigid ryegrass-wheat) was more intense than the intraspecific competition (rigid ryegrass-rigid ryegrass or wheat-wheat).

It has to be noted, that in our study wheat emerged 2–5 days earlier than rigid ryegrass. Consequently, this early emergence of Triticum aestivum allowed its high biomass accumulation, and therefore its competitiveness was high. In Australia, Rerkasem et al. (1980) found that the competitive ability of rigid ryegrass under field conditions was low when rigid ryegrass emerged later or simultaneously with wheat. Early wheat emergence in comparison with weeds is considered to increase wheat competitive ability as previously reported by Kropff et al. (1992) and O'Donovan et al. (1985).

Our experiments also showed that a density of 2–6 wheat plants pot⁻¹ decreased SDW per plant of rigid ryegrass at different densities (data not shown). As shown in Figures 4, 5, increased wheat density seems to increase the competition against rigid ryegrass populations which may be due to the dense wheat canopy and reduction of the availability of light, nutrients, and moisture for rigid ryegrass (Tanji et al., 1997). In another study and by using different rigid ryegrass densities in a field in Australia, Medd et al. (1981) found that SDW of rigid ryegrass in the presence of 40 or 74 wheat plants m⁻² was twice that obtained in the presence of 200 wheat plants m⁻². These findings agree with the results of our study showing that all densities of rigid ryegrass (2, 4, and 6 plant per pot) reduced SDW of wheat and grain yield per pot⁻¹ (Figures 5, 6).

When populations R-1781, R-2041, and S were sown under competitive situations with wheat, their dry weight and height were significantly affected (Figure 5). SDW of the S biotype confirmed the theoretically anticipated response showing the fitness cost of R-1781, R-2041, biotypes. Dry weight and plant height of R-1781, R-2041, and S biotypes were different from non-competitive situation, being various in all studied proportions (Figures 4A, 5 and Table 4).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.